CURRENT PROJECTS

Mechanisms of axon collateral branching

The formation of axon branches is fundamental to the development of complex neuronal circuitry. Branches formed from the axon shaft allow one axon to contact multiple synaptic partners over a large territory. Indeed, deficits in axon branching are observed in a variety of disease states and branching is affected by injury to the nervous system. Our goal is to understand the signaling and cytoskeletal mechanisms underlying the initiation and maturation of axon collateral branches.

The first step in the formation of axon collateral branches is the initiation of filopodia or lamellipodia from the otherwise quiescent axon shaft. Panel A above shows GFP labeled sensory axons in the living spinal cord of a chicken embryo. Arrowheads denote axonal filopodia. The * denotes the base of a collateral branch and the arrow denotes the tip of the branch. We have previously shown that axonal filopodia are formed from precursors accumulations of axonal actin filaments termed axonal actin patches (B, numbers=sec). Our recent work has revealed that initiation of axonal filopodia is driven by localized microdomains of PIP3, the lipid product of phosphoinositide 3-kinase. The microdomains of PIP3 in turn drive the formation of patches of actin filaments that give rise to filopodia (C, numbers=sec). Thus, PIP3 microdomains are the earliest known signaling event in the axon that leads to the formation of axonal filopodia and ultimately collateral branches.

We are currently investigating the molecular mechanisms downstream of PIP3 microdomains that are involved in the formation of axonal actin patches and the emergence of filopodia from actin patches using a variety of imaging approaches and molecular techniques. Through imaging of actin dynamics in axons in the living spinal cord we have now detected actin patch formation and the emergence of axonal filopodia (not shown) and will use this system to further detail the underlying mechanisms and dynamics.

In order for a filopodium to mature into a collateral branch it must be stabilized by the entry of axonal microtubules. A second major aim of the project is to understand the molecular mechanisms that target and retain microtubules in axonal filopodia.

Collaborators: Dr. Lorene Lanier (U of Minnesota), Dr. Elias Spiliotis (Drexel), Dr. Tatyana Svitkina (U Penn)

Support: NIH

Mechanisms of axon retraction

During development axons are guided to their targets where they then establish synaptic contacts with other neurons. The process of sculpting the array of synaptic contacts made by a single axon with multiple target neurons is not only due to the formation of axon branches (see above), but also by the removal of inappropriately targeted axons and branches through the mechanism of retraction. Furthermore, injury to the nervous system causes severing of axons and the severed axons subsequently retract.  Thus, understanding the mechanisms of axon retraction has the potential to unveil potential therapeutic targets for blocking aspects of the response of the nervous system to injury and thus promote later regeneration.

The axon repellent signal Semaphorin3A (Sema3A) is involved both in the guidance and pruning of axons through retraction.  We have shown that Seam3A induces axon retraction by eliciting the formation of an intra-axonal cytoskeleton characterized by actin filament bundles which then serve as a substratum for myosin II-mediated force generation that drives the retraction of the axon (A). In the context of severing-induced axon retraction, we have demonstrated that injury induces the formation of an accumulation of actin filaments at the site of axon severing (A, panel Ax, arrow), which similar to Sema3A-induced axonal actin bundles serves as the substratum for myosin II-driven axon retraction. We termed the severing induced actin structure the actin "cap". We have developed an in situ model system for studying axon retraction through imaging GFP-expressing axons in the living spinal cord (B). Retracting axons in situ exhibit a bulbous tip, termed the retraction bulb, which can be seen in GFP-expressing axons (B) and axons retracting in the rat spinal cord (C, circled).

We are currently dissecting the mechanisms use by Sema3A and axon severing to induce the formation of axon actin bundles and the cap, respectively. Elucidation of these mechanisms may provide insights into therapeutics for blocking axon retraction.

Collaborators: Dr. John Houle (Drexel), Dr. Tatyana Svitkina (U Penn)

Support: Past support, Craig H. Neilsen Foundation, NIH

Mechanisms of diffuse axonal injury in traumatic brain injury (TBI)

TBI causes diffuse axonal injury (DAI). DAI is characterized by the formation of bead-like swellings along the axon shaft which ultimately lead to a break-down of axonal structure and the loss of connectivity between brain regions. Panel (A) shows axonal beads in the corpus callosum as revealed by accumulation on APP. Our research team has have developed an in vitro model system of axonal injury that reproduces many of the feature of DAI in vivo. Panel (B) shows a time lapse sequence of axonal bead formation in vitro in response to membrane fluid-shear damage. We are currently testing the hypothesis that DAI results from injury-induced mechanoporation of the axonal membrane (i.e., formation of pores), which causes localized disruption of the axonal microtubule array and impairs the axonal transport of organelles. Panel (C) shows the location of beads along an injured axon in vitro and the accompanying localized decreases in the intensity of the axonal microtubule array revealed by staining for microtubules. We are also testing the feasibility of blocking TBI-induced DAI by blocking the injury-induced mechanoporation using membrane sealing agents in vitro and in vivo.

collaborators: Dr. Ken Barbee (Drexel), Dr. Ramesh Raghupathi (Drexel)

Support: NIH

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